A team at the UChicago-affiliated Marine Biological Laboratory has achieved the first gene knockout in a cephalopod using the squid Doryteuthis pealeii—an exceptionally important research organism in biology for nearly a century.
The team used CRISPR-Cas9 genome editing to knock out a pigmentation gene in squid embryos, which eliminated pigmentation in the eye and in skin cells (chromatophores) with high efficiency.
“This is a critical first step toward the ability to knock out—and knock in—genes in cephalopods to address a host of biological questions,” said Rosenthal, a senior scientist at MBL.
Crawford, the first author on the study, is a professor of biology at St. Mary’s College of Maryland and a summer Whitman Center investigator at the MBL.
Cephalopods (squid, octopus and cuttlefish) have the largest brain of all invertebrates, a distributed nervous system capable of instantaneous camouflage and sophisticated behaviors, a unique body plan, and the ability to extensively recode their own genetic information within messenger RNA, along with other distinctive features. These open many avenues for study and have applications in a wide range of fields, from evolution and development, to medicine, robotics, materials science and artificial intelligence.
The ability to knock out a gene to test its function is an important step in developing cephalopods as genetically tractable organisms for biological research, augmenting the handful of species that currently dominate genetic studies, such as fruit flies, zebrafish and mice.
It is also a necessary step toward having the capacity to knock in genes that facilitate research, such as genes that encode fluorescent proteins that can be imaged to track neural activity or other dynamic processes.
“CRISPR-Cas9 worked really well in Doryteuthis; it was surprisingly efficient,” said Rosenthal. Much more challenging was delivering the CRISPR-Cas system into the one-celled squid embryo, which is surrounded by an exceedingly tough outer layer, and then raising the embryo through hatching. The team developed micro-scissors to clip the egg’s surface and a beveled quartz needle to deliver the CRISPR-Cas9 reagents through the clip.
Studies with Doryteuthis pealeii have led to foundational advances in neurobiology, beginning with description of the action potential (nerve impulse) in the 1950s, a discovery for which Alan Hodgkin and Andrew Huxley became Nobel Prize laureates in 1963. For decades D. pealeii has drawn neurobiologists from all over the world to the MBL, which collects the squid from local waters.